TDP-43 (TAR-DNA-binding protein of 43 kDa, Official Symbol; TARDBP, NM_007375, NP_031401) is a DNA/RNA-binding protein expressed in almost all types of cells. This protein is predominantly localized in nuclei and known to regulate splicing reaction of RNAs and also participate in the formation of certain microRNAs. Moreover, some of the TDP-43 proteins are known to be transported to cytoplasm and function as carriers for a local transport of RNAs within a cell (Non-Patent Document 1).
TDP-43 has been one of the most noteworthy proteins in the field of neurodegenerative diseases since TDP-43 was found to be a principal component of ubiquitin-positive intracellular inclusions appearing in a lesion site-specific manner in frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) in 2006. FTLD is progressive dementia caused by neurodegeneration of the frontal lobe and/or the temporal lobe. ALS is a progressive motor neuron disease caused by selective degeneration of upper and/or lower motor neurons. The finding of TDP-43 in both FTLD-U and ALS suggests a possibility that these two neurodegenerative diseases, which differ in degeneration site and symptom, share a common mechanism of pathogenesis.
The TDP-43 proteins within the inclusions are aggregated resulting from an abnormal phosphorylation and ubiquitination, and various cytotoxicities are known to occur during the process for aggregation. Cells having the inclusions lack the TDP-43 protein in the nuclei, suggesting that a loss of function of TDP-43 also contributes to the pathogenesis of these diseases.
Accordingly, neurodegenerative diseases accompanying a disappearance of TDP-43 protein from the nuclei and an aggregation thereof in the cytoplasm are categorized as TDP-43 proteinopathy (Non-Patent Document 1).
Thereafter, ALS families having TDP-43 gene mutations were found, and it has been shown that the overexpression of such variant TDP-43 in a mouse nervous system induces neurodegeneration (Non-Patent Document 2). Furthermore, in transgenic mice into which a genomic fragment of human wild-type TDP-43 gene had been introduced, TDP-43 was aggregated in particular neurons and a decline in cognitive functions and impaired motor functions were induced, even though the expression level of TDP-43 was increased in all types of cells (Non-Patent Document 3).
In short, it has been shown that not only the mutant TDP-43 proteins but also the wild-type TDP-43 protein can cause ALS- or FTLD-like pathological conditions when expressed at excessive levels.
It has been further revealed that the concentration of TDP-43 protein in the cerebrospinal fluid of sporadic ALS patients is significantly higher than that of healthy individuals in general (Non-Patent Document 4). There is also a report about increased TDP-43 mRNA levels in autopsied spinal cord tissues of sporadic ALS patients (Non-Patent Document 5).
From these findings, the overexpression of TDP-43 protein is considered to be a primary cause of TDP-43 proteinopathy. In order to treat this disease, energetic studies have been made on methods for correcting the overexpression of TDP-43 protein.
Non-Patent Document 5 has reported that bosutinib and nilotinib approved by the Food and Drug Administration (FDA) as therapeutic drugs for chronic myelocytic leukemia suppress neurodegeneration in transgenic mice overexpressing TDP-43 and ameliorate cognitive functions and motor functions. These drugs are known as blood-brain barrier-permeable tyrosine kinase inhibitors and considered to exert these effects by promoting the clearance of the TDP-43 protein via ubiquitin ligase Parkin.
Non-Patent Document 6 has reported that the clearance of TDP-43 is started when caspase 4, which is a cysteine protease localized on the endoplasmic reticulum membrane, performs cleavage between amino acids at positions 174 and 175 of the TDP-43 protein.
However, these methods are to decrease the amount of the TDP-43 protein in cells by promoting the degradation of the TDP-43 protein. Taking into consideration the fact that the TDP-43 protein itself is cytotoxic in TDP-43 proteinopathy, a method capable of suppressing the production of the TDP-43 protein is expected to produce higher therapeutic effects.
From this point of view, the present inventors established an iPSC (induced pluripotent stem cell) line from ALS patients having TDP-43 gene mutations and established a drug screening system using motor neurons generated from the iPSC by inducing the differentiation (ALS-MN). The ALS-MN is an excellent cell model of the disease that possesses various features (such as poor neurite outgrowth, high vulnerability to stress, and formation of TDP-43-positive aggregates) found in the motor neurons of ALS patients. The present inventors investigated drugs involved in RNA metabolism using this screening system and found that anacardic acid, known as a noncompetitive inhibitor of HAT (histone acetyltransferase), has activities of decreasing a level of TDP-43 mRNA and ameliorating the pathological properties (Patent Document 1, Non-Patent Document 7).
Meanwhile, methods using low-molecular nucleic acids such as antisense oligonucleotides, siRNAs, or microRNAs are known as methods for suppressing certain gene expression specifically. Particularly, microRNAs are greatly expected as drugs since they are endogenous molecules and less likely to cause immune response.
A microRNA forms a miRNA-RISC complex and binds to 3′UTR of a particular mRNA, and causes the degradation or translational inhibition of the mRNA, leading to suppression of its expression. A large number of microRNAs targeting genes involved in various diseases have been identified so far, and drugs containing the microRNAs or low-molecular nucleic acids targeting the microRNAs (anti-miRs) as active ingredients have been developed (e.g., Patent Documents 2 to 4).
However, a microRNA targeting TDP-43 has not yet been reported. Although a large number of microRNAs whose expression varies in TDP-43 proteinopathy have been identified, a microRNA capable of directly regulating the expression of TDP-43 still remains to be discovered.
The TDP-43 protein is known to bind directly to 3′UTR of its own mRNA to promote its degradation as well as a typical microRNA (Non-Patent Document 8). Accordingly, as for TDP-43 gene, a possibility is suggested that the TDP-43 protein itself, instead of microRNAs, regulates its mRNA and protein levels. In addition, it is concerned that a TDP-43-targeting microRNA, if any, might not sufficiently function in cells in which TDP-43 protein has been already overexpressed.
Under these circumstances, developing a method for treating TDP-43 proteinopathy using a microRNA has been considered to be difficult.